Glycosyltransferases (GTs) constitute a large family with approximately 23,000 predicted or known GT sequences in the CAZY database divided into 87 families based upon amino acid similarity. Despite the vast range of GT sugar donors and acceptors (sugars, proteins, nucleic acids, lipids, and small molecules), GTs are generally classified into two simple groups based upon mechanism (inverting or retaining), and primarily fall within two main structural superfamilies (GT-A and GT-B). Lairson L L, et al. (2004) Chem Commun 2243-8; Hu Y., et al. (2002) Chem Biol 9: 1287-96. The GT-B fold is the predominate fold of natural product GTs and is characterized by two closely associated Rossman-like domains, each of which is usually distinguished as the acceptor- and donor-binding domains (N and C-terminal domains, respectively). Despite the wealth of GT structural and biochemical information, attempts to alter GT donor/acceptor specificities via rational engineering have been largely unsuccessful and primarily limited to sequence-guided single site mutagenesis. Hancock S M, et al. (2006) Curr Opin Chem Biol 10: 509-19. While there exists precedent for the directed evolution of carbohydrate-utilizing enzymes, the lack of sensitive high-throughput screens for GTs has also hampered GT directed evolution. Hoffmeister D, et al. (2003) Proc Natl Acad Sci USA 100: 13184-9; Williams G J, et al. (2006) J Am Chem Soc 128: 16238-47.
Nucleotide diphosphate (NDP) sugars are common substrates for GTs where they routinely act as glycoside donors. A generic structure for an NDP sugar is depicted in FIG. 1. In general, NDP sugars represent a class of compounds routinely utilized in the investigation of polysaccharide formation for basic metabolism, intra- and extracellular transport, cell wall biosynthesis within virulent organisms, and drug discovery. However, synthesis of sugar-nucleotides is currently expensive, difficult and time-consuming, and is further complicated by their low solubility in organic solvents and susceptibility to both chemical and enzymatic hydrolysis. Further, an exemplary GT reaction utilizing an NDP sugar, in this case GtfD, is shown in FIG. 2.
While classical synthetic strategies to access sugar nucleotides are available, most require many steps and often suffer from low-yielding reactions, difficult purifications, and a lack of stereochemical control. Accordingly, a need exists for new reagents and routes to provide NDP sugars for a variety of uses in the biomedical field.